Differentiating homozygous, heterozygous and wild-type alleles using a multiplexed hybridization-mediated assay

ABSTRACT

Described are methods of assay design and assay image correction, useful for multiplexed genetic screening for mutations and polymorphisms, including CF-related mutants and polymorphs, using an array of probe pairs (in one aspect, where one member is complementary to a particular mutant or polymorphic allele and the other member is complementary to a corresponding wild type allele), with probes bound to encoded particles (e.g., beads) wherein the encoding allows identification of the attached probe. The methods relate to avoiding cross-hybridization by selection of probes and amplicons, as well as separation of reactions of certain probes and amplicons where a homology threshold is exceeded. Methods of correcting a fluorescent image using a background map, where the particles also contain an optical encoding system, are also disclosed.

RELATED APPLICATIONS

This application is a continuation of application U.S. Ser. No.10/847,046, filed May 17, 2004, which claims priority to U.S.Provisional Application No. 60/470,806, filed May 15, 2003, priority toboth of which is hereby claimed.

BACKGROUND

The standard method of genomic analysis for mutations and polymorphisms,including for CF, is the “dot-blot” method. Samples including targetstrands are spotted onto a nitrocellulose, support, and then contactedwith labeled probes complementary to the mutations or polymorphicregions. The labels allow detection of probe hybridization toimmobilized complementary target sequences, as unbound labeled probesare removed by washing. In another method—a “reverse dot-blot format”—anarray of oligonucleotide probes is bound to a solid support, and thencontacted with a sample including target sequences of interest. See,e.g., U.S. Pat. No. 5,837,832.

Both methods of assaying mutations or polymorphisms have significantdisadvantages. The dot-blot method is itself labor-intensive. It canalso yield erroneous results due to the inaccurate reading of assaysignals, usually done by autoradiography, which adds further labor, asthe probes must be frequently re-labeled. The method described in U.S.Pat. No. 5,837,832 involves a complex and costly on chip synthesis of anarray of oligonucleotides, an approach which is better-suited forlarge-scale genomic analysis and is neither practical nor cost-effectivefor diagnostic applications requiring only a limited but changing numberof probes.

An assay method suitable for multiplexed analysis which avoids many ofthe problems associated with the above methods involves use of randomencoded arrays of microparticles, where the encoding indicates theidentity of an oligonucleotide probe molecule bound thereto. See U.S.patent application Ser. No. 10/204,799: “Multianalyte Molecular AnalysisUsing Application-Specific Random Particle Arrays.” The bead array iscontacted with labeled amplicons, generated from a patient sample, andthe labels are then detected (if the labels are fluorescent, thedetection can be with optical means) and the bound amplicons areidentified by decoding of the array.

In a multiplexed hybridization assay, cross-hybridization amongmismatched, but closely homologous, probes and amplicons can generatefalse positive signals. Thus, the assay should be designed to minimizesuch effects. A number of mutations and polymorphisms are significantonly if they are homozygous, and therefore, to be useful in such cases,the assay must be capable of discriminating heterozygotes fromhomozygotes. Also, in determining the assay results, where both theencoding method for the beads and the determination of assay results iswith optically detectable means, the encoding on the beads can causespectral leakage, which can be affect the assay signal discrimination. Amethod of correcting for such spectral leakage is also needed.

Cystic fibrosis (“CF”) is one of the most common recessive disorders inCaucasians, with an occurrence of 1 in 2000 live births in the UnitedStates. Mutations in the cystic fibrosis (CF) transmembrane conductanceregulator (CFTR) gene are associated with the disease. The number ofCFTR mutations is growing continuously and rapidly, and more than 1,000mutations have been detected to date. See Kulczycki L. L., et al.(2003), Am J Med Genet 116:262-67. Population studies have indicatedthat the most common CF mutation, a deletion of the 3 nucleotides thatencode phenylalanine at position 508 of the CFTR amino acid sequence(designated ΔF508), is associated with approximately 70% of the cases ofcystic fibrosis. This mutation results in the failure of an epithelialcell chloride channel to respond to cAMP (Frizzell R. A. et al. (1986)Science 233:558-560; Welsh, M. J. (1986) Science 232:1648-1650.; Li, M.et al. (1988) Nature 331:358-360; Quinton, P. M. (1989) Clin. Chem.35:726-730). In airway cells, this leads to an imbalance in ion andfluid transport. It is widely believed that this causes abnormal mucussecretion observed in CF patients, and ultimately results in pulmonaryinfection and epithelial cell damage. A number of mutations areassociated with CF, and researchers continue to reveal new mutationsassociated with the disease. The American College of Medical Genetics(“ACMG”) has recommended a panel of 25 of the most common CF-associatedmutations in the general population, especially those in AshkenaziJewish and African-American populations. A multiplexed hybridizationassay for CF-associated mutations in the general population would testfor this panel.

SUMMARY

Described are practical and cost-effective methods of assay design andassay image correction, useful for multiplexed genetic screening formutations and polymorphisms, including CF-related mutants andpolymorphs, using an array of probe pairs (in one aspect, where onemember is complementary to a particular mutant or polymorphic allele andthe other member is complementary to a corresponding wild type allele),with probes bound to encoded particles (e.g., beads) wherein theencoding allows identification of the attached probe. The design methodsdisclosed herein were used to design an assay for CF-related mutationsby hybridization-mediated multiplexed analysis, and were extensivelyvalidated in many patient samples, and demonstrated to be capable ofidentifying the most common mutations, including mutations in exons 3,4, 5, 7, 9, 10, 11, 13, 14b, 16, 18, 19, 20, 21 and introns 8, 12, 19 ofthe CFTR gene.

Before hybridization, the region of interest in the genomic sample isamplified with two primers, one for each strand in the region ofinterest. Of the two strands generated in the PCR amplification step,one is arbitrarily designated herein as “sense” and one as “anti-sense.”In certain instances, it is desirable to select, for subsequent mutationanalysis by hybridization, either the sense target strand (to behybridized to sense probes) or the anti-sense target strand—to behybridized to anti-sense probes. Strand selection is accomplished, forexample, by post-PCR digestion of a phosphorylated strand. Inparticular, strand switching is desirable whenever probe-targetcombinations (e.g., sense-probe/sense target hybridization) involving astable mismatch configuration, such as a G-T base pairing, can beavoided.

Also disclosed are methods of selecting probes and amplicons for geneticscreening for mutations and polymorphisms. The method of selectingprobes and amplicons involves the following steps:

-   providing a family of single-stranded MP amplicons in which one    strand is designated sense and the complementary strand is    designated anti-sense, said MP amplicons including amplified    segments of the genome on which said genetic mutations or    polymorphisms are located;-   selecting complementary MP probes for each member of said family of    MP amplicons;-   examining the degree of homology between either the complementary MP    probes or between the family of MP amplicons;-   dividing said MP probes into one or more probe sets, and dividing    said MP amplicons into sets such that the members of each amplicon    set are complementary to the members of one probe set, said division    based on avoiding homology greater than an acceptance level between    probes in the same set or between MP amplicons in the same set;-   performing for each said set of amplicons in turn, the following    steps for each MP amplicon in said set, in succession:-   (a)(i) determining whether, upon contacting a sense MP amplicon with    a probe set which includes a complementary MP probe to said sense    amplicon, the degree of cross-hybridization of said sense MP    amplicon with other MP probes in the probe set will exceed an    acceptance level; and, if not:-   (a)(ii) retaining said sense MP amplicon in the amplicon set and the    complementary MP probe in the probe set, and repeating step (a)(i)    for another MP amplicon in said family;-   (b)(i) but if said degree of cross-hybridization does exceed said    acceptance level: replacing, in the probe set, the cross-hybridizing    MP probe with the complementary anti-sense MP probe, and replacing,    in the amplicon set, the complementary sense MP amplicon with the    anti-sense MP amplicon complementary to said anti-sense MP probe,    and-   (b)(ii) repeating step (a)(i) and if the degree of    cross-hybridization is within the acceptance level: retaining said    anti-sense MP probe and corresponding complementary anti-sense MP    amplicon in their respective sets and repeating step (a)(i);-   (b)(iii) but if the degree of cross-hybridization exceeds the    acceptance level after repeating step (a)(i): determining whether,    upon contacting said anti-sense MP amplicon with the MP probes in    any other set, the degree of cross-hybridization is within the    acceptance level, and if so, placing the anti-sense MP probe    complementary to said anti-sense MP amplicon into said set and    placing said anti-sense MP amplicon into the set of complementary    anti-sense MP amplicons; but if the degree of cross-hybridization    exceeds the acceptance level following such determination for each    existing probe set, reverting to the original sense MP probe and    complementary sense MP amplicon and placing said sense MP probe and    said complementary sense MP amplicon each into a new set, and-   (c) repeating steps (a) to (c) for another sense MP amplicon in said    family.

Also disclosed is a method for design of pairs of probes (with a memberrespectively complementary to a mutant and a wild type amplicon) forhybridization to labeled amplicons generated by amplification of samplesand wild type controls. For each anticipated variant, probes areprovided in pairs, with one member complementary to the wild typesequence and the other to the variant sequence, the two sequences oftendiffering by only one nucleotide. One method to enhance the reliabilityof hybridization-mediated multiplexed analysis of polymorphisms (hMAP)is to determine the ratio of the signals generated by the capture of thetarget matched and mismatched probes and to set relative ranges ofvalues indicative of normal and heterozygous or homozygous variants.

The method set forth above for selecting probes and amplicons forgenetic screening for mutations and polymorphisms, can be included aspart of a method to select probe pairs (wild-type and variant), byincluding the following steps in the afore-described method:

-   providing a family of single-stranded WT amplicons in which one    strand is designated sense and the complementary strand is    designated anti-sense, said family representing respective amplified    segments of a wild type genome which corresponds to each of the    amplified segments of the genome which was amplified when producing    the family of MP amplicons;-   providing and selecting a sense or anti-sense WT probe so as to have    both a sense WT probe and a corresponding sense MP probe in the same    probe set or, or an anti-sense WT probe and a corresponding    anti-sense MP probe in the same probe set;    determining: (i) whether the degree of cross-hybridization between a    MP amplicon and a corresponding WT probe in a probe set, and between    a WT amplicon and a corresponding MP probe in a probe set, will    exceed the acceptance level and, if so, (ii) determining whether    said degree of cross-hybridization will fall within the acceptance    level if the selected sense or anti-sense MP and WT probes are    replaced with the complementary WT and MP probes; and if so, (iii)    determining whether said complementary WT and MP probes will exceed    the acceptance level for cross-hybridization with amplicons    complementary to other members of the same probe set, and if    so, (iv) determining whether placing the complementary WT and MP    probes into another probe set will exceed the acceptance level for    cross-hybridization with amplicons complementary to other members of    the same probe set, and if not: retaining the complementary WT and    MP probes in said probe set; but if so, (v) repeating step (iv) for    each existing probe set, and if said acceptance level is exceeded    for each existing probe set, placing the complementary WT and MP    probes into a new set and placing the complementary WT and MP    amplicons into a corresponding new set.

Cross-hybridization is a concern in any assay involving multiplexedhybridization, and methods to avoid its deleterious effects on assayresults are included herein. One method to correct forcross-hybridization in an array format, is to set a series oftemperature increments, selected such that at each temperature,probe-target complexes containing particular mismatch configurationswill denature, while those containing matched (“complementary”) basepair configurations will remain intact. The signals generated bycaptured labeled strands hybridized to probes in the array are thenmonitored and recorded at each temperature set point. Analysis of theevolution of differential signals as a function of temperature allowscorrection for each mismatch expected to become unstable above a certain“melting” temperature. After all set points for all mismatches aredetermined, data gathered at lower temperatures can be corrected for allmismatches.

In another aspect, because the assay method herein relies on encodedbeads to identify the probe(s) attached thereto, and the encoding in oneembodiment is by way of dye staining, the assay signals are oftenproduced by using fluorescent labels and removing backgroundcontributions. Specifically, a method of correcting the assay image isdisclosed. That is, within the spectral band selected for the recordingof the assay image, the recorded set of optical signatures produced bytarget capture to bead-displayed probes in the course of the assay arecorrected for the effects of “spectral leakage” (a source of spuriouscontributions to the assay image from the residual transmission) ofintensity emitted by bead-encoding dyes of lower wavelength. An assaydesign is provided herein in which a negative control bead is includedin the random encoded array for each type of encoded bead that producesunacceptably large spectral leakage, for example, for beads containingdifferent amounts of specific encoding dyes.

In the examples described herein, negative control beads display an18-mer C polynucleotide in order to serve a secondary purpose, i.e., topermit correction of assay images for the effects of non-specificadsorption. Preferably, the background correction is performed byconstructing a background map based on the random locations of each typeof negative control bead, where each such type of negative control beadis included in the array at a pre-selected abundance. For each type ofnegative control bead within the array, a background map is generated bylocating the centroids of the beads of that type, constructing theassociated Voronoi tessellation by standard methods (as illustrated inFIG. 3; see, e.g., Seul, O'Gorman & Sammon, “Practical Algorithms forImage Analysis,” Cambridge University Press, 2000; at page 222;incorporated by reference) and then filling each polygon which includesa bead with the intensity of such bead to produce a map (see, e.g., themap shown in FIG. 3). Optionally, standard filtering operations may beapplied to smooth the map; that is, to average out effects fromneighboring pixels. See, e.g., Seul, O'Gorman & Sammon, “PracticalAlgorithms for Image Analysis,” Cambridge University Press, 2000 fordescription of a filter).

Such a map represents a finite sample of the entire backgroundcontributions to the assay image in a manner that accounts for certainnon-linear optical effects associated with arrays composed of refractivebeads, which effects are especially pronounced when the beads are placedinto mechanical traps on a substrate surface. In addition, backgroundmaps will indicate non-uniformities in the background which may arise,for example, from non-uniform illumination or non-uniform distributionof target or analyte placed in contact with the bead array. Maps fornegative control beads of different types, i.e., containing differentamounts of encoding dyes and producing different degrees of spectralleakage, may be normalized to the same mean intensity and superimposedto increase the sampling rate.

The assay image may be corrected as follows by employing the backgroundmap. In certain instances, the map is simply subtracted from the assayimage to produce a corrected assay image. In other embodiments, thebackground can be combined with a “flat fielding” step (See, e.g., Seul,O'Gorman & Sammon, “Practical Algorithms for Image Analysis,” CambridgeUniversity Press, 2000). In this procedure, the constant (i.e., thespatially non-varying) portions of the background map and assay imageare subtracted, and the corrected assay image is divided by thecorrected background map to obtain a “flat fielded” intensity map.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of hybridization of 29 different CFTRmutations; where the smaller open bars represent mutant hybridization,and where hybridization to the “normal” is represented by the largerblack bars (e.g., EX-10 has a high degree of mutant hybridization).

FIG. 2 shows the results of hybridization of 29 CFTR mutations, with themutations being different from those shown in FIG. 1.

FIG. 3 shows a background map of negative control carriers forcorrecting array images.

DETAILED DESCRIPTION

Provided herein are methods for hybridization-mediated multiplexedanalysis of polymorphisms (hMAP) of a designated set of designatedmutations in he Cystic Fibrosis Transmembrane Conductance Regulator(CFTR) gene.

Probes used in the detection of mutations in a target sequence hybridizewith high affinity to amplicons generated from designated target sites,when the entire amplicon, or a subsequence thereof, is fullycomplementary (“matched”) to that of the probe, but hybridize with alower affinity to amplicons which have no fully complementary portions(“mismatched”). Generally, the probes of the invention should besufficiently long to avoid annealing to unrelated DNA target sequences.In certain embodiments, the length of the probe may be about 10 to 50bases, or preferably about 15 to 25 bases, and more preferably 18 to 20bases.

Probes are attached, via their respective 5′ termini, using linkermoieties through methods well known in the art, to encodedmicroparticles (“beads”) having a chemically or physicallydistinguishable characteristic uniquely identifying the attached probe.Probes are designed to capture target sequences of interest contained ina solution contacting the beads. Hybridization of target to the probedisplayed on a particular bead produces an optically detectablesignature. The optical signature of each participating bead uniquelycorresponds to the probe displayed on that bead. Prior to, or subsequentto the hybridization step, one may determine the identity of the probesby way of particle identification and detection, e.g., by decoding orusing multicolor fluorescence microscopy.

The composition of the beads includes, but is not limited to, plastics,ceramics, glass, polystyrene, methylstyrene, acrylic polymers,paramagnetic materials, thoria sol, carbon graphite, titanium dioxide,latex or cross-linked dextrans such as sepharose, cellulose, nylon,cross-linked micelles and Teflon. See “Microsphere Detection Guide” fromBangs Laboratories, Fishers Ind. The particles need not be spherical andmay be porous. The bead sizes may range from nanometers (e.g., 100 nm)to millimeters (e.g., 1 mm), with beads from about 0.2 micron to about200 microns being preferred, more preferably from about 0.5 to about 5micron being particularly preferred.

In certain embodiments, beads may be arranged in a planar array on asubstrate prior to the hybridization step. Beads also may be assembledon a planar substrate to facilitate imaging subsequent to thehybridization step. The process and system described herein provide ahigh throughput assay format permitting the instant imaging of an entirearray of beads and the simultaneous genetic analysis of multiple patientsamples.

The array of beads may be a randomly encoded array, that is, the codeassociated with each bead, placed during assembly into a position withinthe array that is not known a priori, indicates the identity ofoligonucleotide probes attached to said beads. Random encoded arrays maybe formed according to the methods and processes disclosed inInternational Application No. PCT/US01/20179, incorporated herein byreference.

The bead array may be prepared by employing separate batch processes toproduce application-specific substrates (e.g., chip at the wafer scale)to produce beads that are chemically encoded and attached tooligonucleotide probes (e.g., at the scale of about 10⁸ beads/100 μlsuspension). These beads are combined with a substrate (e.g., siliconchip) and assembled to form dense arrays on a designated area on thesubstrate. In certain embodiments, the bead array contains 4000 of 3.2μm beads has a dimension of 300 μm by 300 μm. With different size beads,the density will vary. Multiple bead arrays can also be formedsimultaneously in discrete fluid compartments maintained on the samechip. Such methods are disclosed in U.S. application Ser. No.10/192,352, entitled: “Arrays of Microparticles and Methods ofPreparation Thereof,” which is incorporated herein by reference. Beadarrays may be formed by the methods collectively referred to as“LEAPS™”, as described in U.S. Pat. Nos. 6,251,691, 6,514,771; 6,468,811all of which are also incorporated herein by reference.

Substrates (e.g., chips) used in the present invention may be a planarelectrode patterned in accordance with the interfacial patterningmethods of LEAPS by, e.g., patterned growth of oxide or other dielectricmaterials to create a desired configuration of impedance gradients inthe presence of an applied AC electric field. Patterns may be designedso as to produce a desired configuration of AC field-induced fluid flowand corresponding particle transport. Substrates may be patterned on awafer scale by invoking semiconductor processing technology. Inaddition, substrates may be compartmentalized by depositing a thin filmof a UV-patternable, optically transparent polymer to affix to thesubstrate a desired layout of fluidic conduits and compartments toconfine fluid in one or several discrete compartments, therebyaccommodating multiple samples on a given substrate.

The bead arrays may be prepared by providing a first planar electrodethat is in substantially parallel to a second planar electrode(“sandwich” configuration) with the two electrodes being separated by agap and containing a polarizable liquid medium, such as an electrolytesolution. The surface or the interior of the second planar electrode ispatterned with the interfacial patterning method. The beads areintroduced into the gap. When an AC voltage is applied to the gap, thebeads form a random encoded array on the second electrode (e.g.,“chip”). And, also using LEAPS, an array of beads may be formed on alight-sensitive electrode (“chip”). Preferably, the sandwichconfiguration described above is also used with a planar light sensitiveelectrode and another planar electrode. Once again, the two electrodesare separated by a gap and contain an electrolyte solution. Thefunctionalized and encoded beads are introduced into the gap. Uponapplication of an AC voltage in combination with a light, the beads forman array on the light-sensitive electrode.

In certain embodiments, beads may be associated with a chemically oroptically distinguishable characteristic. This may be provided, forexample, by staining beads with sets of optically distinguishable tags,such as those containing one or more fluorophore or chromophore dyesspectrally distinguishable by excitation wavelength, emissionwavelength, excited-state lifetime or emission intensity. The opticallydistinguishable tags made be used to stain beads in specified ratios, asdisclosed, for example, in Fulwyler, U.S. Pat. No. 4,717,655 (Jan. 5,1988). Staining may also be accomplished by swelling of particles inaccordance with methods known to those skilled in the art, (Molday,Dreyer, Rembaum & Yen, J. Mol Biol 64, 75-88 (1975); L. Bangs, “Uniformlatex Particles, Seragen Diagnostics, 1984). For example, up to twelvetypes of beads were encoded by swelling and bulk staining with twocolors, each individually in four intensity levels, and mixed in fournominal molar ratios. Alternatively, the methods of combinatorial colorencoding described in International Application No. PCT/US 98/10719,incorporated herein by reference, can be used to endow the bead arrayswith optically distinguishable tags. In addition to chemical encoding,beads may also be rendered magnetic by the processes described inInternational Application No. WO 01/098765.

In addition to chemical encoding of the dyes, the beads having certainoligonucleotide primers may be spatially separated (“spatial encoding”),such that the location of the beads provide certain information as tothe identity of the beads placed therein. Spatial encoding, for example,can be accomplished within a single fluid phase in the course of arrayassembly by invoking LEAPS to assemble planar bead arrays in any desiredconfiguration in response to alternating electric fields and/or inaccordance with patterns of light projected onto the substrate.

LEAPS creates lateral gradients in the impedance of the interfacebetween silicon chip and solution to modulate the electrohydrodynamicforces that mediate array assembly. Electrical requirements are modest:low AC voltages of typically less than 10V_(pp) are applied across afluid gap of typically 100 μm between two planar electrodes. Thisassembly process is rapid and it is optically programmable: arrayscontaining thousands of beads are formed within seconds under electricfield. The formation of multiple subarrays, can also occur in multiplefluid phases maintained on a compartmentalized chip surface.

Subsequent to the formation of an array, the array may be immobilized.For example, the bead arrays may be immobilized, for example, byapplication of a DC voltage to produce random encoded arrays. The DCvoltage, set to typically 5-7 V (for beads in the range of 2-6 μm andfor a gap size of 100-150 μm) and applied for <30 s in “reverse bias”configuration so that an n-doped silicon substrate would form the anode,causes the array to be compressed to an extent facilitating contactbetween adjacent beads within the array and simultaneously causes beadsto be moved toward the region of high electric field in immediateproximity of the electrode surface. Once in sufficiently closeproximity, beads are anchored by van der Waals forces mediating physicaladsorption. This adsorption process is facilitated by providing on thebead surface a population of “tethers” extending from the bead surface;polylysine and streptavidin have been used for this purpose.

In certain embodiments, the particle arrays may be immobilized bychemical means, e.g, by forming a composite gel-particle film. In oneexemplary method for forming such gel-composite particle films, asuspension of microparticles is provided which also contain allingredients for subsequent in-situ gel formation, namely monomer,crosslinker and initiator. The particles are assembled into a planarassembly on a substrate by application of LEAPS, e.g., AC voltages of1-20 V_(p-p) in a frequency range from 100's of hertz to severalkilohertz are applied between the electrodes across the fluid gap.Following array assembly, and in the presence of the applied AC voltage,polymerization of the fluid phase is triggered by thermally heating thecell ˜40-45° C. using an infra-red (IR) lamp or photometrically using amercury lamp source, to effectively entrap the particle array within agel. Gels may be composed of a mixture of acrylamide and bisacrylamideof varying monomer concentrations from 20% to 5%(acrylamide:bisacrylamide=37.5:1, molar ratio), or any other lowviscosity water soluble monomer or monomer mixture may be used as well.Chemically immobilized functionalized microparticle arrays prepared bythis process may be used for a variety of bioassays, e.g., ligandreceptor binding assays.

In one example, thermal hydrogels are formed using azodiisobutyramidinedihydrochloride as a thermal initiator at a low concentration ensuringthat the overall ionic strength of the polymerization mixture falls inthe range of ˜0.1 mM to 1.0 mM. The initiator used for the UVpolymerization is Irgacure 2959®(2-Hydroxy-4′-hydroxyethoxy-2-methylpropiophenone, Ciba Geigy,Tarrytown, N.Y.). The initiator is added to the monomer to give a 1.5%by weight solution.

In certain embodiments, the particle arrays may be immobilized bymechanical means. For example, an array of microwells may be produced bystandard semiconductor processing methods in the low impedance regionsof the silicon substrate. The particle arrays may be formed using suchstructures by, e.g., utilizing LEAPS mediated hydrodynamic andponderomotive forces are utilized to transport and accumulate particleson the hole arrays. The AC field is then switched off and particles aretrapped into microwells and thus mechanically confined. Excess beads areremoved leaving behind a geometrically ordered random bead array on thesubstrate surface.

Substrates (e.g., chips) can be placed in one or more enclosedcompartment, permitting interconnection. Reactions can also be performedin an open compartment format similar to microtiter plates. Reagents maybe pipetted on top of the chip by robotic liquid handling equipment, andmultiple samples may be processed simultaneously. Such a formataccommodates standard sample processing and liquid handling for existingmicrotiter plate format and integrates sample processing and arraydetection.

Encoded beads can also be assembled, but not in an array, on thesubstrate surface. For example, by spotting bead suspensions intomultiple regions of the substrate and allowing beads to settle undergravity, assemblies of beads can be formed on the substrate. In contrastto the bead arrays formed by LEAPS, these assemblies generally assumedisordered configurations of low-density or non-planar configurationsinvolving stacking or clumping of beads thereby preventing imaging ofaffected beads. However, the combination of spatial and color encodingattained by spotting mixtures of chemically encoded beads into amultiplicity of discrete positions on the substrate still allowsmultiplexing.

In certain embodiments, a comparison of an assay with a decoded image ofthe array can be used to reveal chemically or physically distinguishablecharacteristics, and the elongation of probes. This comparison can beachieved by using, for example, an optical microscope with an imagingdetector and computerized image capture and analysis equipment. Theassay image of the array is taken to detect the optical signature thatindicates the probe elongation. The decoded image may be taken todetermine the chemically and/or physically distinguishablecharacteristics that uniquely identify the probe displayed on the beadsurface. In this way, the identity of the probe on each particle in thearray may be identified by a distinguishable characteristic.

Image analysis algorithms may be used in analyzing the data obtainedfrom the decoding and the assay images. These algorithms may be used toobtain quantitative data for each bead within an array. The analysissoftware automatically locates bead centers using a bright-field imageof the array as a template, groups beads according to type, assignsquantitative intensities to individual beads, rejects “blemishes” suchas those produced by “matrix” materials of irregular shape in serumsamples, analyzes background intensity statistics and evaluates thebackground-corrected mean intensities for all bead types along with thecorresponding variances. Examples of such algorithms are set forth inInternational Application No. WO 01/098765.

The probe hybridization may be indicated by a change in the opticalsignature, e.g., of the beads associated with the probes. This can bedone using labeling methods well known in the art, including direct andindirect labeling. In certain embodiments, fluorophore or chromophoredyes may be attached to one of the nucleotides added during the probehybridization, such that the probe hybridization to its target changesthe optical signature of beads (e.g., the fluorescent intensitieschange, thus providing changes in the optical signatures of the beads).

Described herein are methods and compositions to conduct accuratepolymorphism analysis for highly polymorphic target regions. Analogousconsiderations pertain to designs, compositions and methods ofmultiplexing PCR reactions.

The density of polymorphic sites in highly polymorphic loci makes itlikely that designated probes directed to selected polymorphic sites,when annealing to the target subsequence proximal to the designatedpolymorphic site, will overlap adjacent polymorphic sites. That is, anoligonucleotide probe, designed to interrogate the configuration of thetarget at one of the selected polymorphic sites, and constructed withsufficient length to ensure specificity and thermal stability inannealing to the correct target subsequence, will align with othernearby polymorphic sites. These interfering polymorphic sites mayinclude the non-designated selected sites as well as non-selected sitesin the target sequence.

The design of covering probe sets is described herein in connection withhybridization-mediated multiplexed analysis of polymorphisms in thescoring of multiple uncorrelated designated polymorphisms, as in thecase of mutation analysis for CF carrier screening. In this instance,the covering set for the entire multiplicity of mutations containsmultiple subsets, each subset being associated with one designated site.In the second instance, the covering set contains subsets constructed tominimize the number of probes in the set, as elaborated herein.

Arrays of bead-associated probes can be used in thehybridization-mediated analysis of a set of mutations within the contextof a large set of non-designated mutations and polymorphisms in theCystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Each ofthe designated mutations in the set is associated with the disease andmust be independently scored. In the case of a point mutation, twoencoded probes are provided to ensure alignment with the designatedsite, one probe complementary to the wild-type, the other to the mutatedor polymorphic target sequence.

In certain embodiments, the identification of the specific targetconfiguration encountered in the non-designated sites is of no interestso long as one of the sequences provided in the covering probe setmatches the target sequence sufficiently closely—and thus matches thetarget sequence exactly to ensure hybridization. In such a case, all orsome of the covering probes may be assigned the same code; in apreferred embodiment, such probes may be associated with the same solidsupport (“probe pooling”). Probe pooling reduces the number ofdistinguishable solid supports required to represent the requisitenumber of probes. In one particularly preferred embodiment, solidsupports are provided in the form of a set or array of distinguishablemicroparticles which may be decoded in situ. Inclusion of additionalprobes in the covering set to permit identification of additionalpolymorphisms in the target region is a useful method to elucidatehaplotypes for various populations.

Suitable probes may be designed to correspond to the known alleleswithin the CFTR gene locus. A number of polymorphisms and mutant allelesare known and available from literature and other sources.

Standard methods of temperature control are readily applied to set theoperating temperature of, or to apply a preprogramed sequence oftemperature changes to, single chips or to multichip carriers. Whencombined with the direct imaging of entire arrays of encoded beads asprovided in the READ™ format of multiplexed analysis, the application ofpreprogrammed temperature cycles provides real-time on-chipamplification of elongation products. Given genomic, mitochondrial orother DNA, linear on-chip amplification eliminates the need forpre-assay DNA amplification such as PCR, thereby dramatically shorteningthe time required to complete the entire typing assay. Time-sensitiveapplications such as cadaver typing are thereby enabled. Moreimportantly, this approach will eliminate the complexities of PCRmultiplexing, a limiting step in many genetic screening and polymorphismanalyses. In a preferred embodiment, a fluidic cartridge provides forsample and reagent injection, as well as temperature control.

The designs, compositions and methods described herein also pertain tothe multiplexed amplification of nucleic acid samples. In a preferredembodiment, covering sets of PCR primers composed of priming andannealing subsequences are used for target amplification.

Described below is a series of steps for selecting an appropriate arrayof probes and targets for hybridization analysis.

Task:

Identify (“select”) a set of probes, P, to perform one or moreconcurrent, “multiplexed” reactions permitting hybridization-mediatedinterrogation of nucleic acid sequences in order to determine thecomposition at each of a set of designated polymorphic sites, S={S₁, Y,S_(N)} said sites being located on M< or =N nucleic acid strands T:={T₁,Y, T_(M)} (“targets”).

Targets—The collection, T, of targets, {T_(i)=(m_(i), σ_(i)); 1< or =i<or =M}, is generated in a polymerase chain reaction (PCR) using PCRprimers designed to place as many polymorphisms or mutations on eachsingle target under the condition that the target length l_(i) notexceed a preset maximal length, l_(max), and wherein the i-th target,T_(i) of length l_(i) is further characterized by:

-   -   a multiplicity, m_(i),        -   giving the number of said designated polymorphisms (or            mutations),        -   wherein Σ(i=1; i=M) m_(i)=N; and    -   an orientation, σ_(i), wherein        -   σ_(i)=+1 for a sense (“cis”) strand; or        -   σ_(i)=−1 for an anti-sense (“trans”) strand.

Probes—Mutation analysis preferably will involve the interrogation ofeach designated mutation site, S_(k), by hybridization of thecorresponding target to at least two designated interrogation probes,P_(k) ^(N) and P_(k) ^(V), of which at least a first probe, P_(k) ^(N),has a sequence that is complementary to the normal (“wild-type”)composition, and of which at least a second probe, P_(k) ^(V), has asequence that is complementary to a variant (“mutant”) composition. Inthe presence of polymorphisms or mutations at sites within theinterrogated subsequence other than the designated sites, it generallywill be desirable to provide “degenerate” probes matching theanticipated compositions at non-designated sites. The designationP_(k)=P_(k) (S_(k)) hereinafter is understood to refer to all probesdirected to the k-th designated site such that P_(k) is characterized bya number of probes, each of these probes having an orientation, σ_(ik),opposite to that of the cognate target.

Specifically, probes are to be selected, and probe-target reactions areto be configured in a manner involving one or more sets of reactions,each of these reactions being performed in a separate container, in sucha way as to minimize-the interaction of any target subsequencecontaining a designated polymorphic site or mutation, S_(k), with anybut its corresponding designated probes, P_(k).

Strategy—While not necessarily generating an optimal configuration, thefollowing “heuristic” strategy provides the basis for a systematicprocess of assay optimization as a function of critical parametersincluding a maximal acceptable degree of similarity between twosequences, expressed in terms of a homology score, as well as a maximalacceptable level of “cross-hybridization”, manifesting itself inmagnitude of “off-diagonal” elements, P_(i) T_(j) of a co-affinitymatrix (see U.S. application Ser. No. 10/204,799, entitled:“Multianalyte molecular analysis using application-specific randomparticle arrays”) showing the degree of interaction between all probesand all targets in a given group or set.

To minimize cross-hybridization between any given target and probesdirected to other targets, distribute targets and their correspondingprobes—into a number, C, of containers in order to perform C separate“multiplexed” hybridization reactions, said number being chosen to be assmall as possible given a preset maximal acceptable level of sequencesimilarity (“maximal homology score”) between targets in the samecontainer.

To minimize cross-hybridization between any given target and probesdirected to other targets in the same container, switch the orientationof such other targets and that of their corresponding probes, allowingfor the possible reassignment of any target to another, possibly newcontainer.

Certain targets may have more than one region, each having a designatedprobe in the array which hybridizes with it. To minimizecross-hybridization as well as competitive hybridization within the samecontainer in such case, reduce the multiplicity of such an “offending”target by redesigning the PCR primer sets in order to produce two (ormore) smaller targets to replace the original single target, each of thenew targets having a lower multiplicity of hybridization regions thanthe original.

Implementation—The pseudocode below provides a description of theheuristic process of configuring the reaction so as to minimizecross-hybridization.

I - Assign targets - and cognate probes - to c sets (“Containers”) c =0; DO {  REFSEQ = SelectTarget(T); /*  randomly pick a target sequencefrom given collection, T*/  ShrinkCollection (T, 1);   /*  remove selected target from collection */  S = L(c);  InitializeList (L(g),REFSEQ);   /* place selected target into   new   set(“group”)implemented  in  the form of a list, S */  AlignTargets(REFSEQ, T, HScores); /*  align remaining targets to REFSEQ   by pairwise  alignment   or  multiple  sequence   alignment;   return  homology scores */  SortTargets (HScores, T);     /* rank target seq'sin the order of increasing   homology  score  with   respect  to REFSEQ;   first entry least similar,   last entry most similar   toREFSEQ */  t = AssignTargets (maxHSCORE, /*  remove  targets  from  S, ,T); collection  in  the  order  of increasing homology scores up tomaxHScore and place them into  the  list,  starting  at  top; return number  of  targets assigned to the list, S */  ShrinkCollection (T ,t); /*  remove  t  selected targets from collection */  c++; }WHILE ( Tnot EMPTY);Optionally, one or more lists may be pruned should they contain morethan an acceptable number of targets (for example, if it is determined,based on too many targets in a list, that maxHScore should be lowered)by removing targets from the bottom of one or more of the lists andplacing them back into the collection T.

II - Refine group configuration FOR ( i=0; i<c; i++)   /*examine eachgroup in turn */ {   S = L(i);   /* List S holds targets in current  group */   PofS = SelectProbes ( P, S);   /* select from P all probes  directed to targets in current list */   /*  each probe is designed to  match at least one target - this   is referred to as the probe  cognate for that target; NOTE:   refers to diagonal elements in  co-affinity matrix */   WHILE (S not EMPTY)   {     T = PopTarget (S);    PerformProbeTargetRxn (PofS, T)   /* place T in contact with all  selected probes, preferably   arranged in a probe array */     FOR(each probe, P, in PofS)     {       I = DetermineInteractionStrength(P, T);   /* eliminate unacceptably large     off-diagonal element in    co-affinity matrix*/       IF( (P not cognate to T) AND (I > maxI) )      {         FlipOrientation (P);     /* flip probe orientation    */         FlipOrientation (TcP);         /* flip orientation oftarget cognate to P */       }     }     /*  check  “flipped” targets        in list S*/     flippedT = PopTarget (S);    PerformProbeTargetRxn (PofS, flippedT)     /* place T in contact  with  all  selected  probes,   preferably arranged in a probe   array*/     FOR (each probe, P, in PofS)     {       I =DetermineInteractionStrength (P, flippedT);     /*     eliminate    unacceptably large off-     diagonal element in co-     affinitymatrix*/       IF( (P not cognate to flippedT) AND (I > maxI) )       {        PushTarget (flippedT, TempList);       /* Place flipped target      into  temporary list */       }     }   } } S = TempList; /* ListS holds flipped targets in temp list */ /* Check targets in temp list */WHILE (S not EMPTY) {   T = PopTarget (S);   FOR (j=0; j <c; j++)   {    IF( L(j) != L(T) ) /*  Check  T  against  probes  in  all existinglists with exception of those in T's original list */     {       L =L(j);       PofL = SelectProbesFrom List (L);     /*  select  probes  in  Group L */       PerformProbeTargetRxn (PofL, T)     /* place T incontact   with all selected probes,   preferably arranged in a probe  array */       FOR (each probe, P, in PofL)       {         I =DetermineInteractionStrength (P, T);   /* eliminate unacceptably large    off-diagonal element in     co-affinity matrix*/         IF( (P notcognate to T) AND (I > maxI) )         {           FlipOrientation (T));    /* flip target orientation     back to original */          FlipOrientation (PcT); /* flip orientation of p r o b e    cognate to target T */            PushTarget(T, NewList);   /* initnew group */         }       }     }   } }         FlipOrientation (P);    /* flip probe orientation     */         FlipOrientation (TcP);        /* flip orientation of target cognate to P */

EXAMPLE I CFTR Assay

Genomic DNA extracted from several patients was amplified withcorresponding primers in a multiplex PCR (mPCR) reaction. The PCRconditions and reagent compositions were as follows:

PRIMER DESIGN: One of the primers (sense or antisense, depending ondesign considerations, discussed below) was modified with a label (suchas, Cy3, Cy5 and Cy5.5) at the 5′ end and the corresponding primer forthe complementary sequence had a phosphate group added at the 5′ end, sothat the amplicon could be digested by λ exonuclease during post-PCRprocessing of the target (see below). Hybridization was detected bydetection of the dyes (Cy3, Cy5 or Cy5.5) in the hybridized product.Multiplex PCR (mPCR) was performed in two groups with the followingprimers (Tables I and II), and with the reagents and under theconditions listed below. The exon number where the mutation is locatedappears below in the left-had column of Tables I and II.

TABLE I Artificial sequence artificial primer mPCR Group I Primers:(“Cy” denotes a dye label, and “P” denotes a phosphate modification, atthe 5′ end of the primer) EX-5-1-Cy GTC AAG CCG TGT TCT A GAT SEQ IDNO.:1 EX-5-2-P GTT GTA TAA TTT ATA ACA ATA GT SEQ ID NO.:2 EX-7-1-P ACTTC AAT AGC TCA GCC TTC SEQ ID NO.:3 EX-7-2-Cy TAT GGT ACA TTA CCT GTATTT TG SEQ ID NO.:4 EX-9-1-P TGG TGA CAG CCT CTT CTT SEQ ID NO.:5EX-9-2-Cy GAA CTA CCT TGC CTG CTC CA SEQ ID NO.:6 EX-12-1-P TCT CCT TTTGGA TAC CTA GAT SEQ ID NO.:7 EX-12-2-Cy TGA GCA TTA TAA GTA AGG TAT SEQID NO.:8 EX-13-1-P AGG TAG CAG CTA TTT TTA TGG SEQ ID NO.:9 EX-13-2-CyATC TGG TAC TAA GGA CAG SEQ ID NO.:10 EX-14B-1-P TCT TTG GTT GTG CTG TGGCT SEQ ID NO.:11 EX-14B-2-Cy ACA ATA CAT ACA AAC ATA GT SEQ ID NO.:12EX16A-1P CTT CTG CTT ACC ATA TTT GAC SEQ ID NO.:13 EX16A-2-Cy TAAT ACAGAC ATA CTT AAC G SEQ ID NO.:14 EX-18-1-P GG AGA AGG AGA AGG AAG AG TSEQ ID NO.:15 EX18-2-Cy ATC TAT GAG AAG GAA AGA AGA SEQ ID NO.:16Ex-19-1-Cy GGC CAA ATG ACT GTC AAA GA SEQ ID NO.:17 Ex-19-2-P TGC TTCAGG CTA CTG GGA TT SEQ ID NO.:18

TABLE II mPCR Group II Primers: Ex-3-1-Cy C GGC GAT GTT TTT TCT GGA GSEQ ID NO.:19 Ex-3-2-P T ACA AAT GAG ATC CTT ACC C SEQ ID NO.:20Ex-4-1-P AGC TTC CTA TGA CCC GGA TA SEQ ID NO.:21 Ex-4-2-Cy TGT GAT GAAGGC CAA AAA TG SEQ ID NO.:22 EX-10-1-P TGT TCT CAG TTT TCC TGG AT SEQ IDNO.:23 EX-10-2-Cy CTC TTC TAG TTG GCA TGC TT SEQ ID NO.:24 Ex-11-1-P CAGATT GAG CAT ACT AAA AG SEQ ID NO.:25 EX11-2-Cy AC ATG AAT GAC ATT TACAGC SEQ ID NO.:26 Int-19-1-Cy AA TCA TTC AGT GGG TAT AAG C SEQ ID NO.:27Int-19-2-P CCT CCT CCC TGA GAA TGT TGG SEQ ID NO.:28 EX-20-1-P C TGG ATCAGG GAA GA GAA GG SEQ ID NO.:29 EX20-2-Cy TCC TTT TGC TCA CCT GTG GT SEQID NO.:30 EX21-1-P TGA TGG TAA GTA CAT GGG TG SEQ ID NO.:31 EX21-2-CyCAA AAG TAC CTG TTG CTC CA SEQ ID NO.:32

PCR Master mix composition For 20 μl reaction/sample: Components Volume(μl) 10X PCR buffer 2.0 25 mM MgCl₂ 1.4 dNTPs (2.5 mM) 4.0 Primer mix(Multiplex 10x) 3.0 Taq DNA polymerase 0.6 ddH2O 3.0 DNA 6.0 Total 20

PCR Cycling Conditions

Amplifications were performed using a Perkin Elmer 9700 thermal cycler.Optimal primer concentrations were determined for each primer pair. Thereaction volume can be adjusted according to experimental need.

Post PCR processing: Following amplification, PCR products were purifiedusing either a QIAquick PCR purification kit (QIAGEN, Cat # 28104), orby Exonuclease 1 treatment (Amersham). For the latter procedure: analiquot of 8 μl of PCR product was added in a clean tube with 2.5 μl ofExonuclease 1 (Amersham), incubated at 37° C. for 15 minutes anddenatured at 80° C. for 15 min. Thereafter, single stranded DNA wasgenerated as follows:

PCR reaction products were incubated with 2.5 units of λ exonuclease in1× buffer at 37° C. for 20 min, followed by enzyme inactivation byheating to 75° C. for 10 min. Under these conditions, the enzyme digestsone strand of duplex DNA from the 5′-phosphorylated end and releases5′-phosphomononucleotides (J. W. Little, et al., 1967). Single-strandedtargets also can be produced by other methods known in the art, althoughheating the PCR products to generate single stranded DNA, isundesirable. The single stranded DNA can be used directly in the assay.

ON CHIP Hybridization—The CFTR gene sequence from Genebank was used tomodel the wild-type. The 52 probes were divided into two groups on thebasis of their sequence homologies, in accordance with the “heuristic”probe selection algorithm, i.e., in such a way as to avoid overlappinghomologies among different probes to the extent possible. The mutationsincluded in each group were selected so as to minimize overlap betweenprobe sequences in any group and thereby to minimize intra-groupcross-hybridization under multiplex assay conditions.

Probe sequences were designed by PRIMER 3.0 software (see The BroadInstitute website, incorporated herein by reference), seeking to includethe following characteristics in each probe:

-   (a) a mismatch in the center of the probe;-   (b) probe length 16-21 bases;-   (c) low self compatibility;-   (d) 30-60% GC content; and-   (e) no more than three consecutive identical bases.    Each probe sequence was aligned with its complementary exon    sequence. See Baylor College of Medicine HGSC. BCM Search Launcher    website incorporated herein by reference. The percent homology    between each probe and non-desired target sequences (i.e., those    sequences representing mutations other than those which thc probe is    intended to hybridize with) was calculated, and probes were selected    such that the percent homology between probes for each mutation and    non-desired target sequences on the same array was less than 50%.

Probe selection was further refined based on the heuristic selectionalgorithm, set forth above. Probe selection was also refined in part onexperimental selection, and in part on consideration that certainmismatched base pairs, particularly, G-T, will tend to be stable. Ininstances where probes could hybridize incorrectly with mismatchesforming a G-T pairing, and in certain other instances, the anti-senseprobes were used, rather than the sense probes, if such stablemismatches could be avoided, or if it was experimentally demonstratedthat incorrect hybridization was eliminated by using the antisenseprobe. The cases where antisense probes were used are indicated in theProbe Sequence Table III below.

Wild type and mutant probes for 26 CF mutations were synthesized witheither 5′ Biotin-TEG or amine modification at the 5′ end (Integrated DNATechnologies). Different bead chemistry can use a different 5′ end, suchthat a biotin modification is coupled to beads coated with neutravidin,and an amine modification is coupled to beads coated with BSA. Probeswere dissolved in 1× TE or dsH₂O at a concentration of 100 μM. Analiquot of 100 μl of 1% bead solids, for each type of bead, was washedthree times with 500 μl of TBS-1 (1× TE, 0.5 M NaCl2). Probes were addedto 500 μl bead suspension and incubated at room temperature for 45-60minutes on a roller. Beads were washed once with wash solution TBS-T (1×TE, 0.15 M NaCl₂, 0.05% TWEEN 20 (polysorbate detergent)) or PBS-T(Phosphate buffered saline, TWEEN 20 (polysorbate detergent)) and twicewith TBS-2 (1× TE, 0.15 M NaCl₂) and re-suspended in 1× TBS-2. Beadswere assembled on the surface of chips as described earlier. The probeswere also divided into two groups and assembled on two separate chips. Athird group was assembled for reflex test including 5T/7T/9Tpolymorphisms. Negative and positive controls were also included on thechip surface, and assay signal was normalized using these controls. Fornegative controls, beads were coupled with a 10-mer strand of dCTP(Oligo-C) and immobilized on the chip surface. For a positive controlsignal, the human β Actin sequence was used. The signal from Oligo-C wasused as the background to subtract the noise level and β Actin was usedto normalize the data.

TABLE IIIA Hybridization Group I: Bead cluster Mutation 1OLIGO-C(control) 2 BA 3 OligoC-1 4 G85E-WT 5 G85E-M 6 621+1G>T-WT 7621+1G>T-M 8 R117H-WT 9 R117H-M 10 I148-WT 11 I148-M 12 A455E-WT 13A455E-M 14 508-WT 15 OLIGOC-2 16 F508 17 I507 18 G542-WT 19 G542-M 20G551D-WT 21 G551D-M 22 R560-WT 23 R560-M 24 R553X-WT 25 R553X-M 26OLIGOC-3 27 1717−1G>A-WT 28 1717−1G>A-M 29 3849+10kb-WT 30 3849+10kb-M31 W1282X-WT 32 OLIGOC-4 33 W1282X-M 34 N1303K-WT 35 N1303K-M 36OLIGOC-5

TABLE IIIB Hybridization Group II Bead Cluster Mutation 1 BA 21898+5G-WT 3 OLIGO-C(control) 4 1898+5G-M 5 OLIGO-C-1 6 R334W-WT 7R334W-M 8 1898+1G>A-WT 9 1898+1G>A-WT 10 1078delT-M 11 OLIGO-C-2 12D1152-WT 13 D1152-M 14 R347P-WT 15 R347P-M 16 711+1G>T-WT 17 711+1G>T-M18 3659delC-WT 19 3659delC-M 20 OLIGO-C-3 21 R1162X-WT 22 R1162X-M 232789+5G-WT 24 2789+5G-M 25 3120+1G>A-WT 26 3120+1G>A-WT 27 OLIGO-C-4 28A455E-WT 29 A455E-M 30 2184delA-WT 31 2184delA-M 32 1078delT-WT 33OLIGO-C-5

TABLE IIIC Hybridization Group III (total 6 groups) Cluster # Mutation 1β Actin 1 Oligo C 2 5T 3 7T 4 9TProbe sequences for detecting each mutation were as follows (probes tosense or antisense sequences were selected as described above):

TABLE IV NORMAL/VARIANT SEQUENCE CAPTURE PROBES EX-3 AT GTT CTA TGG AATCTT TT TA SEQ ID NO.:33 G85E AT GTT CTA TGA AAT CTT TT TA SEQ ID NO.:34EX-4 TA TAA GAA GGT AAT ACT TC CT SEQ ID NO.:35 621-M TATAA GAA GTTAATACT TC CT SEQ ID NO.:36 INT-4 CC TCA TCA CAT TGG AAT GC AG SEQ IDNO.:37 I148T CC TCA TCA CAC TGG AAT GC AG SEQ ID NO.:38 EX-4 CAA GGA GGAACG CTC TAT CG C SEQ ID NO.:39 R117H CAAGGA GGA ACA CTC TAT CGC SEQ IDNO.:40 EX-5 ATG GGT ACA TAC TTC ATC AA A SEQ ID NO.:41 711 + 1G ATG GGTACA TAA TTC ATC AAA SEQ ID NO.:42 EX-7 GAA TAT TTT CCG GAG GAT GAT SEQID NO.:43 334-M GAA TAT TTT CCA GAG GAT GAT SEQ ID NO.:44 EX-7 CAT TGTTCT GCG CAT GGC GGT SEQ ID NO.:45 347-M CAT TGT TCT GCC CAT GGC GGT SEQID NO.:46 EX-7 CT CAG GGT TCT TTG TGG TG TT SEQ ID NO.:47 1078DEL T CTCAG GGT TC TTG TGG TG TT SEQ ID NO.:48 EX-9 ACA GTT GTT GGC GGT TGC TGGSEQ ID NO.:49 A455E ACA GTT GTT GGA GGT TGC TGG SEQ ID NO.:50 EX-10 AAAGAA AAT ATC ATC TTT GGT SEQ ID NO.:51 F508 AAA GAA AAT ATC ATT GGT GTSEQ ID NO.:52 I507 AAA GAA AAT ATC TTT GGT GT SEQ ID NO.:53 EX-12 ATATTT GAA AGG TAT GTT CT TT SEQ ID NO.:54 1898 + 1 ATA TTT GAA AGA TAT GTTCT TT SEQ ID NO.:55 Ex-13 GAA ACA AAA AAA CAA TCT TTT SEQ ID NO.:56 2184delA GAA ACA AAA AA CAA TCT TTT SEQ ID NO.:57 EX-14B TTG GAA AGT GAG TATTCC ATG SEQ ID NO.:58 2789 + 5G TTG GAA AGT GAA TAT TCC ATG SEQ IDNO.:59 EX-16 ACT TCA TCC AGA TAT GTA AAA SEQ ID NO.:60 31120 + 1G/A ACTTCA TCC AGG TAT GTA AAA SEQ ID NO.:61 Ex-11 TAT AGT TCT TGG AGA AGG TGGSEQ ID NO.:62 G542X TAT AGT TCT TTG AGA AGG TGG SEQ ID NO.:63 EX-11 TCTTTA GCA AGG TGA ATA ACT SEQ ID NO.:64 R560 TCT TTA GCA ACG TGA ATA ACTSEQ ID NO.:65 EX-11-553/551 GAG TGG AGG TCA ACG AGC AAG SEQ ID NO.:66G551D GAG TGG AGA TCA ACG AGC AAG SEQ ID NO.:67 R553X GTG GAG GTC AATGAG CAA GA SEQ ID NO.:68 EX-11 TGG TAA TAG GAC ATC TCC AAG SEQ ID NO.:691717-M TGG TAA TAA GAC ATC TCC AAG SEQ ID NO.:70 EX-18 ACT CCA GCA TAGATG TGG ATA SEQ ID NO.:71 1152X ACT CCA GCA TAC ATG TGG ATA SEQ IDNO.:72 EX-19-SENSE GAA CTG TGA GCC GAG TCT TTA SEQ ID NO.:73 R1162X GAACTG TGA GCT GAG TCT TTA SEQ ID NO.:74 EX-19 TGG TTG ACT TGG TAG GTT TAGSEQ ID NO.:75 3659 TGG TTG ACT TG TAG GTT TAC SEQ ID NO.:76 INT-19 T TAAAAT GGT GAG TAA GA CAC SEQ ID NO.:77 3849 T TAA AAT GGC GAG TAA GA CACSEQ ID NO.:78 EX-20 TGC AAC AGT GGA GGA AAG CCT SEQ ID NO.:79 1282X TGCAAC AGT GAA GGA AAG CCT SEQ ID NO.:80 EX-21 A TTT AGA AAA AAC TTG GAT CCSEQ ID NO.:81 N1303K A TTT AGA AAA AAG TTG GAT CC SEQ ID NO.:82β A-PROBE AG GAC TCC ATG CCC AG SEQ ID NO.:83

The hybridization buffer has been optimized for use in uniplex and/ormultiplex hybridization assays and is composed of (finalconcentrations): 1.125 M Tetramethyl-Ammonium Chloride (TMAC), 18.75 mMTris-HCL (pH 8.0), 0.75 mM EDTA (pH 8.0) and 0.0375% SDS. Ten μl ofhybridization mixture containing buffer and ssDNA was added on the chipsurface and incubated at 55° C. for 15 minutes. This is a shorterhybridization time than the several hours normally used, because longerhybridization times tend to generate uncontrolled excess hybridization.The chip was washed with 1× TMAC buffer three times, covered with aclean cover slip and analyzed using a BAS imaging system. Images areanalyzed to determine the identity of each of the probes. The resultsare shown below in FIGS. 1 and 2.

Each allele of a given mutation was analyzed as follows. First, thesignal from the hybridized alleles was corrected as follows:

-   -   (i) Signal for allele A (labeled amplicon) =Raw signal from        labeled amplicon-hybrid minus raw counts from negative        (background) control    -   (ii) Signal for allele B (unlabeled amplicon) =Raw signal from        unlabeled amplicon-hybrid minus raw counts from negative        (background) control        Then an allelic ratio was calculated:    -   Allelic ratio = Signal for allele A/Signal for allele B

When the value of (i) was less than or equal to zero, it was adjusted to0.01 to avoid the generation of negative values. Allelic ratios of >2were scored as homozygous for allele A (indicating mutant/polymorph),while an allelic ratio of <0.5 was scored as homozygous for allele B(wild type). An allelic ratio of 0.8 to 1.2 was scored as heterozygous.Values which fell in between these thresholds were considered ambiguousand the assay was repeated.

EXAMPLE II Screening of Multiple Patient Samples—Side-by-Side Comparisonof hMAP with Dot Blot Analysis

A number of patient samples were obtained and amplified for simultaneousscreening. The method of amplification and primer design was asdescribed above. After amplification, analysis techniques on sampleswere compared for 26 CFTR mutations. A set was analyzed usingconventional dot blot hybridization methods, and the same set wasanalyzed with the methods and reagents of the invention. The results foreach patient sample were compiled and both results were compared. Therewas 100% concordance with the two methods of detection. The number ofsamples identified as positives for each mutation are listed in Table V.

TABLE V Comparison of Testing of Samples Samples tested by dot-blot andmethods described herein Mutations # Positives # Negatives Total G85E 1111 22 G85E/621+1G 8 8 621+1G>T 11 13 24 621+1G>T/delF508 2 2 R117H 19 19R117H/delF508 1 1 I148T 48 48 delF508 58 14 72 I507 11 11 delF508/R560 11 G542X 44 11 55 G551D 11 11 R553X 15 15 1717−1G>A 14 14 R560T 9 93849+10kbC>T 25 14 39 W1282X 53 13 66 N1303K 31 15 46 mPCR-WT 87 87711+1G>T 19 9 28 711+1G>T/621+1G 1 1 R334W 19 11 30 R347P 13 13 1078delT11 11 A455E 18 11 29 1898+1G>A 24 10 34 2184delA 10 10 20 2789+5G>A 2010 30 3120+1g>A 18 10 28 R1162X 13 8 21 3569delC 8 8 D1152 47 9 56mPCR-WT 80 80 TOTAL 939

It should be understood that the terms, expressions and examplesdescribed herein are exemplary only and not limiting and that processesand methods can be performed in any order, unless the sequence of stepsis specified. The invention is defined only in the claims which followand includes all equivalents of the claims.

1. A method of differentiating homozygous, heterozygous and wild-typealleles in a target sample comprising mutant or polymorphic alleles orwild-type alleles using results obtained from a probe array, whereinindividual probes in the array are designed to detect designated mutantor polymorphic alleles or wild-type alleles through hybridization ofsaid probes and targets, where the results compensate for hybridizationbetween probes and targets other than their designated targets,comprising: amplifying the genomic regions in the target sample that arepredicted to include said mutant or polymorphic alleles or saidwild-type alleles, thereby producing a first set of labeled ampliconshaving a region complementary to a subsequence of said mutant orpolymorphic alleles (if said mutant or polymorphic alleles are presentin said target sample) and a second set of labeled amplicons having aregion complementary to a subsequence of said wild-type alleles (if saidwild-type alleles are present in said target sample); providing an arrayof probe pairs, the first member of each pair being complementary, inwhole or in substantial part, to a subsequence of a first-set ampliconand the second member of each pair being complementary, in whole or insubstantial part, to a subsequence of a second-set amplicon; contactingunder hybridizing conditions said array of probe pairs with saidamplicons; detecting hybridization between probes and said amplicon setsbased on the presence of signals from the labeled, hybridized amplicons,said signals being corrected to adjust for hybridization between probesand amplicons other than their designated amplicons as follows: (i)determining the intensity of signals from hybridization of first-setamplicons and from hybridization of second-set amplicons, as correctedfor background signals, and (ii) determining the ratio of said signals,wherein the ratio of the intensity of first-set amplicon signals to theintensity of second-set amplicon signals is designated as a first ratio(a), and wherein the ratio of the intensity of second-set ampliconsignals to the intensity of first-set amplicon signals is designated assecond ratio (b); and defining three relative ranges of values for saidratios such that: (i) the lowest range of ratio (a) indicates that saidtarget sample is homozygous for wild-type alleles and the lowest rangeof ratio (b) indicates that said target sample is homozygous for mutantor polymorphic alleles, (ii) a middle range of either ratio (a) or (b)indicates that said target sample is heterozygous, and (iii) the highestrange of ratio (a) indicates that the said target sample is homozygousfor mutant or polymorphic alleles and the highest range of ratio (b)indicates that said target sample is homozygous for wild-type alleles.2. The method of claim 1 further including the step of generatingsingle-stranded DNA from said amplicons.
 3. The method of claim 2further including the step of labeling one of the strands of either ofsaid amplicon sets.
 4. The method of claim 1 wherein said relative valueranges are defined such that: >2 indicates that said target sample ishomozygous for mutant or polymorphic alleles, <0.5 indicates that saidtarget sample is homozygous for wild-type alleles and 0.8 to 1.2indicates that said target sample is heterozygous.